Trace Elements in the Large Population-Based HUNT3 Survey.

The Nord-Trøndelag Health Study (The HUNT Study) is a large health survey population study in the county of Trøndelag, Norway. The survey has been repeated four times in about 10-year intervals. In the HUNT3 survey (2006-2008), we collected 28,000 samples for trace element analysis. Blood samples from 758 healthy persons without known occupational exposure were selected for multielement analysis of a small sample of blood (0.25 mL). The aim of the study was to determine the minimum blood volume that can be used for the analytical procedure and to compare our results with previously published results of similar surveys in healthy populations. Samples were digested and the concentration of selected trace elements was determined by ICP-MS. We report results on essential elements (B, Co, Cu, Mn, Se and Zn) as well as non-essential elements (As, Be, Br, Cd, Cs, In, La, Pb, Hg, Nd, Ni, Nb, Pd, Pt, Sm, Ta and Sn). Results are similar to previous studies on the HUNT3 population, and with a few exceptions, our data compares very well with results obtained in recent studies from other countries. We wanted to test a minimum volume of blood in a large-scale analytical program. For a number of nonessential elements, our results were below the limit of detection. We suggest that future studies using similar ICP-MS equipment as analytical tool should use at least 0.5 mL of blood.

Functional and phenotypic differences of pure populations of stem cell-derived astrocytes and neuronal precursor cells.

Availability of homogeneous astrocyte populations would facilitate research concerning cell plasticity (metabolic and transcriptional adaptations; innate immune responses) and cell cycle reactivation. Current protocols to prepare astrocyte cultures differ in their final content of immature precursor cells, preactivated cells or entirely different cell types. A new method taking care of all these issues would improve research on astrocyte functions. We found here that the exposure of a defined population of pluripotent stem cell-derived neural stem cells (NSC) to BMP4 results in pure, nonproliferating astrocyte cultures within 24-48 h. These murine astrocytes generated from embryonic stem cells (mAGES) expressed the positive markers GFAP, aquaporin 4 and GLT-1, supported neuronal function, and acquired innate immune functions such as the response to tumor necrosis factor and interleukin 1. The protocol was applicable to several normal or disease-prone pluripotent cell lines, and the corresponding mAGES all exited the cell cycle and lost most of their nestin expression, in contrast to astrocytes generated by serum-addition or obtained as primary cultures. Comparative gene expression analysis of mAGES and NSC allowed quantification of differences between the two cell types and a definition of an improved marker set to define astrocytes. Inclusion of several published data sets in this transcriptome comparison revealed the similarity of mAGES with cortical astrocytes in vivo. Metabolic analysis of homogeneous NSC and astrocyte populations revealed distinct neurochemical features: both cell types synthesized glutamine and citrate, but only mature astrocytes released these metabolites. Thus, the homogeneous cultures allowed an improved definition of NSC and astrocyte features.

Silver nanoparticle-induced cytotoxicity in rat brain endothelial cell culture.

Silver nanoparticles (AgNPs) are among the most widely commercialised engineered nanomaterials, because of their antimicrobial properties. They are already commonly used in medical devices, household products and industry. Concerns have been raised about potential adverse health effects due to increasing dispersion of AgNPs in the environment. The present study examined the cytotoxic effects of spherical, citrate-coated AgNPs (10, 50 and 100 nm) in rat brain endothelial (RBE4) cells and investigated whether the observed effects can be explained by the intrinsic toxicity of the particles or the silver ions released from the particles. The results indicated that exposure of RBE4 cells to AgNPs lead to significant reduction in dye uptake as measured with the Neutral red (NR) assay. The effect was found to be related to particle size, surface area, dose and exposure time. In contrast, silver ions increased NR uptake (ca. 10%) in RBE4 cells after 1h, while a reduction in NR uptake was observed after 24h exposure at high concentrations (20-30 µM). Colony formation, as an indicator of proliferation ability, was completely inhibited by AgNPs at concentrations higher than 1 µg/ml. Silver ions had less effect on the colony formation of RBE4 cells than AgNPs.

The in vitro effects of Trolox on methylmercury-induced neurotoxicity.

Methylmercury (MeHg), an environmental toxicant primarily found in fish and seafood poses a dilemma to both consumers and regulatory authorities given the nutritional benefits of fish consumption vs. possible adverse neurological damage caused by MeHg. The present study addresses whether supplementation with 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox), alters the neuro-oxidative effects of MeHg in C6-glioma and B35-neuronal cell lines. As indicators of cytotoxicity, reduced glutathione (GSH), reactive oxygen species (ROS) and mitochondrial activity (MTT) were measured. The cellular mercury (Hg) content was measured with high resolution-inductively coupled plasma mass spectrometry (HR-ICPMS). The amount of MeHg-induced ROS was significantly reduced (p<0.05) after treatment with 50muM Trolox in C6 glial cell line. However, treatment with Trolox did not induce any significant increase in GSH levels or MTT activity in either of the cell lines. In addition, treatment with Trolox did not induce any significant changes in intracellular MeHg levels. The MeHg and Trolox treated C6 glial cell line differed significantly (p<0.05) from the B35 cell line for MTT, ROS and GSH activity. These findings provide experimental evidence that preincubation with Trolox prevents MeHg-induced ROS generation in C6 glial cell line by quenching of free radicals and not by changes in intracellular GSH or MeHg content.

Caenorhabditis elegans metallothioneins protect against toxicity induced by depleted uranium.

Depleted uranium (DU) is a dense and heavy metal used in armor, ammunition, radiation shielding, and counterbalances. The military usage has led to growing public concern regarding the health effects of DU. In this study, we used the nematode, Caenorhabditis elegans, to evaluate the toxicity of DU and its effects in knockout strains of metallothioneins (MTs), which are small thiol-rich proteins that have numerous functions, such as metal sequestration, transport, and detoxification. We examined nematode viability, the accumulation of uranium, changes in MT gene expression by quantitative reverse transcription-PCR, and the induction of green fluorescent protein under the control of the MT promoters, following exposure to DU. Our results indicate that (1) DU causes toxicity in a dose-dependent manner; (2) MTs are protective against DU exposure; and (3) nematode death by DU is not solely a reflection of intracellular uranium concentration. (4) Furthermore, only one of the isoforms of MTs, metallothionein-1 (mtl-1), appears to be important for uranium accumulation in C. elegans. These findings suggest that these highly homologous proteins may have subtle functional differences and indicate that MTs mediate the response to DU.

The in vitro effects of selenomethionine on methylmercury-induced neurotoxicity.

Selenium (Se) has been reported to reduce the severity of MeHg-induced neurological deficits. Therefore, we investigated whether 24h. preincubation or 50min. coincubation with selenomethionine (SeMet) was effective in reducing methylmercury (MeHg)-induced cytotoxicity in C6-glioma and B35-neuronal cell lines. As indicators of cytotoxicity, reduced glutathione (GSH), reactive oxygen species (ROS) and mitochondrial activity (MTT) was assessed. Measurement of GSH with the fluorescent indicator MCB-monochlorobimane indicated that in SeMet preincubated C6 cells, MeHg treatment resulted in a significant (p<0.001) decrease in GSH levels as compared to coincubation group. Treatment with SeMet did not induce any significant changes in MTT activity in either of the cell lines as compared to the MeHg group. However, the amount of MeHg-induced ROS was significantly reduced (p<0.001) after SeMet preincubation in both the cell lines. The intracellular Se content was measured with high resolution-inductively coupled plasma mass spectrometry (HR-ICPMS). In both the cell lines the intracellular Se levels increased after pre- and coincubation with 20 and 50microM SeMet. However, the preincubation group exhibited increased Se content in both the cell lines and varied (p<0.001) from coincubation group. These differences in the Se content were maintained after 10microM MeHg treatment for 50min. In C6-gliomas, the cell associated-MeHg measurements using (14)C-labeled MeHg indicated a significant increase (p<0.001) in MeHg content in preincubated cells as compared to coincubated cells. These findings provide experimental evidence that preincubation with SeMet increases Se content in cells and prevents against increased MeHg-induced ROS generation.

Minerals and trace elements in commercial infant food.

Adequate nutrition during infancy is essential for lifelong health and wellbeing. Breast-feeding is highly recommended for the first six month of life, but from then on complementary feeding is necessary. Industrially produced food is an important part of the diet for many infants and toddlers in developed countries. We have determined the concentration of major minerals and trace elements in 76 different products of infant formula, porridges, fruit purée and dinners by HR-ICP-MS. The products were collected from three suppliers on the Norwegian market (Nestlé, Tine and Nyco Pharma). A daily menu was also composed and the estimated daily intake was compared with current recommendations. All products were within the upper tolerable limit for the minerals of trace elements analysed and toxic elements were present at very low levels. A diet based solely on industrially prepared food products will provide a sufficient intake of minerals and trace elements.

Separation of proteins including metallothionein in cerebrospinal fluid by size exclusion HPLC and determination of trace elements by HR-ICP-MS.

A method to study the protein binding patterns of trace elements in human cerebrospinal fluid (CSF) is described. Proteins in CSF samples were separated by size exclusion chromatography combined with high performance liquid chromatography (SEC-HPLC). The column was calibrated to separate proteins in the molecular weight range 6-70 kDa. Fractions were then analyzed off-line for trace elements using high resolution inductively coupled plasma mass spectrometry (HR-ICP-MS). We were able to accurately determine more than 10 elements of clinical interest in the CSF fractions. Results are presented for Cd, Mn, Fe, Pb, Cu and Zn. The total concentrations of 16 trace elements in human plasma and CSF are also presented. The method was able to differentiate the relative contribution of metallothionein and other proteins towards metal binding in human CSF.

Brain accumulation of depleted uranium in rats following 3- or 6-month treatment with implanted depleted uranium pellets.

Depleted uranium (DU) is used to reinforce armor shielding and increase penetrability of military munitions. Although the data are conflicting, DU has been invoked as a potential etiological factor in Gulf War syndrome. We examined regional brain DU accumulation following surgical implantation of metal pellets in male Sprague-Dawley rats for 3 or 6 mo. Prior to surgery, rats were randomly divided into five groups: Nonsurgical control (NS Control); 0 DU pellets/20 tantalum (Ta) pellets (Sham); 4 DU pellets/16 Ta pellets (Low); 10 DU pellets/10 Ta pellets (Medium); 20 DU pellets/0 Ta pellets (High). Rats were weighed weekly as a measure of general health, with no statistically significant differences observed among groups in either cohort. At the conclusion of the respective studies, animals were perfused with phosphate-buffered saline, pH 7.4, to prevent contamination of brain tissue with DU from blood. Brains were removed and dissected into six regions: cerebellum, brainstem (pons and medulla), midbrain, hippocampus, striatum, and cortex. The uranium content was measured in digested samples as its 238U isotope by high-resolution inductively coupled plasma-mass spectrometry. After 3 mo postimplantation, DU significantly accumulated in all brain regions except the hippocampus in animals receiving the highest dose of DU (p < 0.05). By 6 mo, however, significant accumulation was measured only in the cortex, midbrain, and cerebellum (p < 0.01). Our data suggest that DU implanted in peripheral tissues can preferentially accumulate in specific brain regions.